Method and a preparation to select for transfected DNA in mammalian cells

ABSTRACT

The invention provides methods and products equal in a selection system for determining the presence or absence of a vector in a mammlian cell. The vector of the invention carries and is capable of expressing a gene encoding a protein capable of effecting the conversion of a precursor to an essential amino acid. A mammalian cell line is created with conditions capable of causing the vector to be introduced into the mammalian cell line. Then, the created mammalian cell line is grown in medium containing a precursors to the essential amino acid but not containing the growth-supporting amount of the essential amino acid and those cells capable of growing are selected.

TECHNICAL FIELD

This invention generally relates to the fields of molecular biology andbiochemistry and more specifically relates to methods and productsuseful in determining whether a vector has been introduced into amammalian cell line.

BACKGROUND OF THE INVENTION

The ability to introduce foreign genes into mammalian cells has provento be a powerful tool both in the research laboratory and for thecommercial production of biologicals. Foreign genes are commonlyintroduced either on plasmid or viral vectors or by co transfection withsuch vectors. Several different methods have been used to introduceforeign genes including calcium phosphate precipitation, protoplastfusion, electroporation and viral infection. For many combinations ofvectors, transfection methods, and mammalian cell lines, stabletransfection is a rare event. Methods for the selection of cellscontaining foreign DNA are usually necessary. Therefore, selectablemarkers have been incorporated into many vectors.

There are a number of different selective methods for transfected DNA inmammalian cells. These methods may be divided into two classes based onthe conferred phenotype. The first class consists of selection relatingto dominant drug resistant markers. Markers in this class includeresistance to neomycin (G418) (Southern and Berg, 1982), hygromycin B(Sugden et al., 1985) and mycophenolic acid (Mulligan and Berg, 1985).The second class consists of selection relating to complementation of anauxotrophic mutation. Several examples of this approach include thethymidine kinase (tk) gene (Wigler et al., 1977), the hypoxanthineguanine phosphoribosyl transferase (hgprt) gene (Mulligan and Berg,1985) and the dihydrofolate reductase gene (dhfr) (Kaufman and Sharp,1982).

The selective systems mentioned above have found broad applicability tothe introduction of foreign DNA into a variety of mammalian cell types.However, there are limitations to each of the individual approaches. Forexample, some cell lines are natively resistant to G418: others growpoorly in mycophenolic acid even in the presence of the vector; andstill others have a reduced growth rate under hygromycin B selection.Selection methods using neomycin and hygromycin B require highconcentrations (greater than 100 ug/ml) and therefore are relativelyexpensive. Hygromycin B is also unstable in cell culture mediumnecessitating frequent refeeding in order to maintain selection.

Selection methods relying upon the complementation of auxotrophicmutations also have drawbacks. In contrast to dominant marker systems,further modification to the recipient cell is required. These systemsalso require the isolation of rare tk deficient, hgprt deficient or dhfrdeficient cells This limits these approaches to cell lines where suchmutants already exist or cell lines with high cloning efficiencies suchthat appropriate mutants may be isolated.

Aside from the drawbacks of known selective systems, it is sometimesnecessary to independently introduce several different genes into asingle cell line. Therefore, it is desirable to have several independentselective systems.

A new selective approach should have the following properties.Preferably, the selective marker is not normally present in therecipient cell line. Therefore, the recipient cell line does not have tobe modified by, for example, mutation to remove a native property, andthe usefulness of the marker does not depend upon the ability to createsuch a mutation. Most preferably the selective marker is not present inmammalian cells so that the selective system may be used in connectionwith mammalian cells. Further, the gene encoding the selective markerpreferably is relatively small in order to facilitate introduction intovectors with limited "available space." The products used in theselective system should be stable in cell culture medium. Finally, theproducts used in the selective system should be relatively inexpensive.These and other objects are achieved according to the invention.

SUMMARY OF THE INVENTION

The invention relates to a method for selecting for a vector introducedinto mammalian cells. Mammals are not capable of synthesizing some aminoacids which are referred to as essential amino acids. Because theycannot synthesize the essential amino acids, mammalian cells in culturewill not grow unless these amino acids are supplied in the growthmedium. However, other organisms such as bacteria are capable ofsynthesizing these amino acids from a suitable precursor(s). Accordingto the invention, a non-mammalian gene(s) which encodes an enzyme(s)involved in the non-mammalian biosynthesis of an essential amino acid isincorporated into a vector. This gene(s) is incorporated into the vectorin such a way that it can be expressed when the vector is introducedinto a host mammalian cell.

Thus, the invention provides a method for selecting for mammalian cellscontaining a vector. First, the vector is provided. The vector carriesand is capable of expressing in mammalian cells a gene, the expressionof which gene results in a protein capable of effecting the conversionof the precursor to an essential amino acid. The mammalian cell linethen is treated with conditions capable of causing the vector to beintroduced into the mammalian cell line. Then, the cells containing thevector are selected by growing the treated cells in the absence of agrowth-supporting, exogenous supply of the essential amino acid but inthe presence of the precursor. Only those cells containing the vectorwill be capable of growing under such conditions.

Preferably, the gene product is an enzyme and the treated cells aregrown in medium containing a precursor to the essential amino acid, butnot containing a growth supporting amount of the essential amino acid.In this manner, the expression of the gene carried on the vector willresult in the production of an enzyme which will convert the precursorinto the essential amino acid thereby allowing the cells to grow.Preferably the precursor is selected from the group consisting ofα-keto-β-methyl valeric acid, α-ketoisocaproic acid, meso-a,ε-diaminopimelic acid, saccharopine, homocystene, cystathionine,phenylpyruvic acid, homoserine phosphate, homoserine, indole-3-glycerolphosphate, α-ketovaleric acid and histidinol.

Preferably, the vector is carrying and capable of expressing a geneencoding an enzyme involved in the synthesis of an amino acid selectedfrom the group consisting of arginine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, threonine, tryptophan and valine.Preferably the enzyme is selected from the group consisting ofα-keto-β-methyl valeric acid transaminase, α-ketoisocaproic acidtransaminase, meso-α,ε-diaminopimelic acid decarboxylase, phenylpyruvicacid transaminase, α-ketoisovaleric acid transaminase, threoninesynthetase, tryptophan synthetase and histidinol dehydrogenase.

Most preferably, the vector contains the E. coli histidinoldehydrogenase (HDH) gene. In E. coli and other micro organisms, theenzyme HDH catalyzes the final step in the biosynthesis of histidine,the conversion of histidinol to histidine. Histidine is an essentialamino acid for mammalian cells. In the preferred vector, the bacterialHDH gene is promoted by the Herpes Simplex Virus (HSV) thymidine kinase(tk) gene promoter and is joined to the HSV tk 3' polyadenylation signalwhich allows the expression of the HDH gene in mammalian cells. When thehost mammalian cells are transfected with the vector, cells which takeup and express the vector DNA can be selected for by growth in culturemedium which does not contain the essential amino acid but does containthe suitable precursor of the essential amino acid. Only those cellscontaining the vector and expressing the HDH gene will grow.

An example of the selection medium of the invention is a mediumessentially equivalent to RPMI 1640, but containing histidinol insteadof histidine.

The invention also provides a mammalian cell or mammalian cell linetransfected with the vector of the invention.

These and other aspects of the invention will be better understood withreference to the drawings and to a particular example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the reaction catalyzed by histidinol dehydrogenase.

FIG. 2 depicts the published restriction map of the E. coli his operonfragment and the observed restriction map of the pHis17 isolate whichcontains substantially the same fragment.

FIG. 3 is a schematic map of pMF6, a vector carrying HSV-tk derivedregulatory sequences, and into which the HDH gene was introduced.

FIG. 4 depicts the growth curve of control cells and cells treated witha plasmid containing the HDH and hyg® genes.

FIG. 5 depicts the growth curve of cells containing the plasmid pAS1 inmedium containing hygromycin or histidinol.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following embodiment of the invention is based on the histidinoldehydrogenase (HDH) gene and its ability to confer growth in mediumcontaining histidinol, but not containing histidine. HDH catalyzes thefinal step in the biosynthesis of histidine in bacterial cells and othermicro-organisms. The reaction catalyzed by HDH is diagrammed in FIG. 1.Histidine is an essential amino acid for mammalian cells and thisnutritional requirement is typically supplied by the cell culture media.HDH activity is not present in mammalian cells.

According to the invention, a vector expressing HDH when introduced intomammalian cells allows mammalian cells to metabolize histidinol tohistidine. This provides a basis for selection of transfectants if themedium contains histidinol and is substantially free of histidine. Inaddition, histidinol is a reversible inhibitor of protein synthesis inhuman cells (Hansen et al., 1972) in the absence of substantial amountsof histidine. This inhibition by histidinol of protein synthesis stillis present even in the presence of low levels of histidine (present byway of the serum, for example). The mechanism of inhibition of proteinsynthesis appears to be mediated through histidinol substitution forhistidine on the tRNA which then blocks protein elongation. Thus,according to the invention, two levels of selection are provided. First,the provision of the histidine required to support cell growth andsecond, the removal of the inhibitory histidinol from the medium.

HDH functions as a multimer with identical subunits (Burger et al.,1979). The apparent molecular weight of the active species in S.typhimurium is 83,000 daltons. The HDH gene has been cloned from E. coliand the nucleotide sequence has been reported (Bruni et al., 1980;Chiariotti et al, 1986). The HDH gene contains 1,305 base pairs andencodes a protein with an approximate molecular mass of 22,700 daltons.The HDH gene is sufficiently small to allow for its incorporation intomany vector systems. For comparison purposes, the genes which conferresistance to neomycin or hygromycin B are each approximately 1000 bp.

MATERIALS AND GENERAL PROCEDURES Materials

Restriction endonucleases were obtained from New England Biolabs,Beverly Mass. and were used according to the manufacturer'srecommendations. Horse serum was obtained from Hazelton Inc., Denver,Pa. Hygromycin B was obtained from Calbiochem, LaJolla, Calif. Luriabroth contains 10gm/l Bacto-tryptone, 5gm/l Bacto yeast extract, and10gm/l NaCl and was obtained from Difco Laboratories, Detroit, Mich. Allother medium, medium components and chemicals were obtained from SigmaChemical Company, St. Louis, Mo. The L3 human lymphoblastoid cell lineis a derivative of the AHH-1 cell line (Crespi and Thilly, 1984). TheAHH-1 cell line may be substituted for L3 cells in the proceduresoutlined herein. AHH-1 is available from the ATCC, Rockville, Md., USAunder accession No. CRL 8146. Further, the selection system describedherein is intended for use in all mammalian cell lines and the inventionis not limited to the particular cell line used in the example describedbelow. Recombinant DNA procedures were performed according to standardtechniques (Maniatis et al, 1982).

Isolation of the Histidinol Dehydrogenase Gene.

We have isolated a portion of the histidine operon from the standard E.coli strain HB 101. HB 101 is commercially available from BethesdaResearch Lab., Gaithersburg, Md. Our isolation was based on thepublished restriction map for this operon. The HDH gene is containedwith a 5.3 kb Hind III fragment of genomic DNA. E. coli DNA was digestedwith Hind III, fractionated based on molecular weight and fragments of5.3 kb were isolated. The 5.3 kb fragments were introduced into theunique Hind III site of pUC19 a plasmid commercially available fromBethesda Research Laboratories, Gaithersburg, Md. Forty-five recombinantplasmids were screened for restriction sites characteristic of thedesired fragment. Each plasmid was first screened for Pst I restrictionssites. Promising candidate plasmids were then screened for Bgl II andHpa I restriction sites. One isolate designated pHis17 was found to havea restriction map identical to that reported in the literature(Chiariotti et al, 1986). A restriction map of our isolate pHIS17, alongwith the published restriction map reported in the literature, ispresented in FIG. 2. Data for Ava II sites are available for pHis17only. The pUC19 DNA of the pHis17 isolate is not shown.

Introduction of the Histidinol Dehydrogenase Gene into a Vector.

The next step was the introduction of the HDH gene into a vector systemwhich would allow HDH expression in mammalian cells. For this study weperformed additional restriction mapping in order to identify a meansfor relatively efficient excision of the HDH gene from pHis17.

Additional restriction mapping indicated that the enzymes Ava II andHind III were relatively efficient at excising the HDH gene from thepHis17 isolate (FIG. 2). Ava II cut the isolate approximately 50 bp 5'to the HDH gene and Hind III cut the isolate approximately 600 bp 3' tothe HDH gene. The resulting fragment was approximately 2 kb long.

The Ava II/Hind III fragment was rendered blunt ended and modified bythe addition of Sal I linkers. The modified fragment was introduced intothe pMF6 expression vector. The pMF6 expression vector is a modificationof the pHEBo vector developed by Sugden et al, (1985). The pHEBo vectorwas obtained from Dr. Sugden of the University of Wisconsin, Madison,Wis. However, it will be understood by those skilled in the art that awide variety of vectors may be substituted for this and for the othervectors employed.

The vector pHEBo bears the genes for ampicillin resistance andhygromycin resistance, and contains sequences from the origin ofreplication of the Epstein-Barr virus which allow it to replicateautonomously in Epstein-Barr virus-transformed lymphoblastoid cells(Sugden et al, 1985). pHEBo possesses unique sites for the enzymes ClaI, Hind III, Bam HI and Sal I, which may be used for the insertion ofthe DNA of interest. An expression vector derived from pHEBo andcontaining the HSV tk gene 5' and 3' controlling sequences flanking anunique Sal I site was constructed. Again, those skilled in the art willrecognize that many other controlling sequences may be substituted.

To introduce the 3' poly A addition signal, the plasmid pHSV106(McKnight et al, 1980a), bearing the HSV tk gene, was digested with PvuII and Xho I linkers were added. The pHSV106 was obtained from BethesdaResearch Labs, Bethesda, Md. The DNA was then digested with Sma I andSal I linkers were attached. This resulted in a 0.6 kb fragment bearingthe 3' poly A additional signals of the HSV tk (McKnight et al, 1980b;Wagner et al, 1981) which could be inserted into the Sal I site ofpHEBo. pHEBo DNA was digested with Sal I and treated with alkalinephosphatase to prevent recircularization of pHEBo during the ligationreaction. Of the two possible orientations, the fragment would becorrectly inserted when the preserved Sal I site at the 5' end of thefragment was proximal to the Bam HI site of pHEBo, and the nonfuctional3' Sal I-Xho I fusion site was distal to the Bam HI site. The correctplasmid, designated p12L, was then cut with Bam HI and Sal I. HSV tk 5'promoter sequences were isolated by cutting pHSV106 with Bgl II,repairing the ends and adding Sal I linkers. The pHSV106 DNA was cutwith Bam HI and the resulting 0.7 kb fragment was inserted into the BamHI/Sal I cut p12L. The resulting plasmid, designated pMF6, bears both 5'and 3' controlling sequences from the HSV tk gene and preserved theunique Sal I site for the insertion of genes to be expressed.

FIG. 3 is a schematic map of pMF6. The thin line represents pBR322sequences, the black box represents EVB sequences, the open boxes areHSV-tk-derived regulatory sequences for the hyg® gene which is thehatched box. The stippled and double hatched boxes represent the 5' and3' sequences of the HSV-tk gene respectively. Restriction sites areindicated as follows: Bam HI, B; Cla I, C; Eco RI, E; Hind III, H; NarI, N; Sal I, S; X/S denotes the junction formed by ligation of Xho I andSal I ends

After introduction of the HDH gene into pMF6, the orientation of theinsert was verified by restriction analysis. The construct containingthe HDH gene in the proper orientation was designated pAS1.

Transfection.

The plasmid pAS1 was introduced into the L3 human lymphoblast line viaprotoplast fusion (Yoakum et al., 1984). E. coli HB 101 containing theplasmid pAS1 were grown in Luria broth containing 50 ug/ml ampicillin(150 ml total volume). When the bacteria concentration yielded a OD₆₀₀of 0.6, 200 ug/ml chloramphenicol was added to the culture and theculture was stirred overnight. Bacteria were centrifuged 2000×g for 10minutes and the cell pellet resuspended in 1.5 ml of HBS-20 (20 mMHEPES, 20% sucrose, pH7.1) and 0.48 ml of a 10 mg/ml lysozyme solution(in HBS-20, filter sterilized) was added. The bacteria were incubated atroom temperature for 45 minutes. The lysozyme reaction was terminated bythe addition of 0.24 ml of 1.25M CaCl₂. Excess Ca++ was chelated byadding 0.6 ml of 0.25M EDTA. The above protoplast preparation wasdiluted by the addition of 6 ml of HBS-9 (20 mM HEPES, 9% sucrose, pH7.1).

L3 cells (2×10⁷ cells) were centrifuged and the cell pellet dispersed bytapping the tube. 1.5 ml of PEG-fusion reagent (48% [w/v] polyethyleneglycol purified according to Yoakum et al, [1984] with the balance RPMImedium 1640) was added to the cells. 2.5 ml of the protoplast suspensionwas added immediately and the resulting suspension was centrifuged at800×g for 3 minutes. The pellet was dispersed by tapping the tube, 1.5ml PEG-fusion reagent was added and the cells were incubated for 1minute. The PEG-fusion reagent was diluted with 50 ml of control medium,the cells were then centrifuged and resuspended in 80 ml of controlmedium containing 100 U/ml Penicillin, 100 ug/ml streptomycin and 100ug/ml gentamycin.

Selection medium.

The selection medium was a modification of RPMI 1640, lacked histidineand had the following composition (pH 6.85):

    ______________________________________                                        MEDIUM COMPONENT                                                                              CONCENTRATION (mg/l)                                          ______________________________________                                        Alanine         13.35                                                         Arginine        200                                                           Asparagine      22.50                                                         Aspartic acid   19.95                                                         Cystine         50                                                            Glutamic acid   22.05                                                         Glutamine       300                                                           Glycine         11.25                                                         Hydroxyproline  20                                                            Isoleucine      50                                                            Leucine         40                                                            Lysine-HCl      50                                                            Methionine      15                                                            Phenylalanine   15                                                            Proline         17.25                                                         Serine          15.75                                                         Threonine       20                                                            Tryptophan      5                                                             Tyrosine        20                                                            Valine          20                                                            p-Aminobenzoic acid                                                                           1                                                             Biotin          1                                                             Calcium pantothenate                                                                          1                                                             Choline-HCl     1                                                             Folic acid      1                                                             Glucose         3500                                                          myo-Inositol    2                                                             Niacinamide     1                                                             Phenol red      5.5                                                           Pyridoxine HCl  1                                                             Riboflavin      0.1                                                           Thiamine        1                                                             Vitamin B.sub.12                                                                              0.005                                                         CaCl.sub.2 2H.sub.2 O                                                                         132.5                                                         KCl             200                                                           KH.sub.2 PO.sub.4                                                                             366                                                           MgSO.sub.4      49                                                            NaCl            6070                                                          NaH.sub.2 CO.sub.3                                                                            2000                                                          Na.sub.2 HPO.sub.4                                                                            400                                                           NaH.sub.2 PO.sub.4                                                                            61                                                            ______________________________________                                    

Selected medium also contained varying amounts of histidinol (200-500uM) and was supplemented with 10% horse serum. Control medium had thesame formulation but contained 80 uM histidine in place of thehistidinol.

EXAMPLES Example 1 Selection by colony formation

After protoplast fusion, cells were grown for two days in control mediumbefore beginning selection. The cultures were centrifuged to remove thehistidine-containing medium. The cells were then plated in the selectionmedium at 20,000 cells per well in flat bottom 96 well plates (Corning,Corning, N.Y.) by the procedure of Furth et al (1981).

Histidinol resistant colonies were recovered after 14 days of incubationfrom the cultures treated with pAS1 at a frequency of 1×10⁻⁶. Nohistidinol resistant cells were recovered from control cultures (lessthan 2633 10⁻⁷).

Example 2 Selection in bulk culture

Selections for hygromycin resistant and histidinol-resistant bulkpopulations were performed concurrently with the selection forhistidinol resistant cells via colony formation. Thehygromycin-resistant population was selected by growth in mediumcontaining 80 uM histidine and 400 ug/ml hygromycin B (10% horse serum).The growth curves for plasmid-treated cells and control cells withoutplasmid are presented in FIG. 4. The control cells did not growappreciably in either selective media. Cells treated with plasmid showedgrowth in both hygromycin and histidinol after 15 days in culture.Hygromycin selection and histidinol selection appear to have comparableperformance.

Example 3 Expression of unselected, co-transfected genes

Two aliquots of cells treated with plasmid pAS1 and selected for theirability to grow in medium containing histidinol were centrifuged. Onewas resuspended in medium containing 80 uM histidine, 200 ug/mlhygromycin B and no histidinol. The other was resuspended in mediumcontaining 200 uM histidinol with no histidine (selection medium). Asshown in FIG. 5, the growth in both cultures was equivalent.

Thus the histidinol-resistant population was completely cross resistantto hygromycin B, which was expected given that both functions are on thesame plasmid. This indicates that selection of cells containing the HDHgene by growth in histidinol-containing medium can be used to select forexpression of other foreign genes contained on the same vector. Thoseskilled in the art will recognize that the other foreign gene(s) whoseexpression in the host is desired, need not be on the same vector.Expression of genes which are co-transfected on a piece of DNA separatefrom the selectable gene can also be selected for (Wigler et al, 1979).

Those skilled in the art also will recognize many equivalents to thispreferred embodiment of the invention. The gene catalyzing the last stepin the biosynthesis of any essential amino acid such as tryptophansynthetase could be incorporated into the vector Selection would then beaccomplished by growing the cells in medium lacking the essential aminoacid but containing the precursor for the final step in the biosynthesisof the essential amino acid, such as indole-3-glycerol phosphate in thecase of tyrptophan synthetase. The criteria for a successful systemincludes the expression of the gene, the stability of the precursor inmedium and the ability of the host cell to take up the precursor fromthe medium. More than the final gene for the biosynthetic pathway couldbe included on the vector coupled with selection via growth in thepresence of the appropriate intermediate precursor of the essentialamino acid in place of the immediate precursor. In fact, thesupplementation of the medium with a precursor of the essential aminoacid is not always necessary. If, for example, the entire histidineoperon were transferred, selection could be accomplished merely bygrowth in medium lacking histidine since mammalian cells are capable ofsynthesizing 5-phosphoribosyl 1 pyrophosphate which is the startingmaterial in the biosynthesis of histidine.

It further will be understood by those skilled in the art that mutantmammalian cell lines that require for growth an amino acid normallynonessential also may be used according to the invention. In thisinstance, a vector carrying the gene for that enzyme capable ofeffecting the conversion of a precursor into that normally nonessentialamino acid would be used as a selection tool. Therefore, the term"essential amino acid" may include amino acids that are not produced bythe host mammalian cell line either in that particular cell line'snaturally occurring condition or a mutant condition.

The preferred vector into which the HDH gene is inserted fortransfection into the host cells depends on the type of host to betransfected. In the embodiment described above, a human lymphoblastoidcell line was used as the host. The pMF6 expression vector, which is amodification of the pHEBo vector developed by Sugden et al (1985), is asuitable expression vector for human lymphoblastoid cell lines.Accordingly the HDH gene was inserted into a derivative of pHEBo (pMF6)for transfection into the human lymphoblastoid cells. Other vectors, ofcourse, are suitable for this cell line. Moreover, as is well known tothose skilled in the art, other vectors would be more appropriate forother cell lines.

The preferred medium contains a precursor of an essential amino acid butdoes not contain a growth-supporting amount of that amino acid. Thepreferred selection medium for the above described embodiment is free ofhistidine and contains histidinol. The medium may be prepared and storedas a solution. Alternatively, the medium may be initially prepared as apowder and stored for later dissolution in a liquid such as water orphysiologic saline. The preferred selection medium also depends, inpart, on other characteristics of the host cells to be transfected.Human lymphoblastoid cells grow well in RPMI 1640. Thus, in the exampledescribed above, the composition of the selection medium was similar toRPMI 1640, but contained histidinol instead if histidine. Formulationsbased on other media such as MEM, DMEM or BME would be more appropriate,for example, for anchorage dependent cells.

LITERATURE CITED

Bruni, C. B., Musti, A. M , Frunzio R. and Blasi, F, (1980) J.Bacterial. 142:32.

Burger, E., Gorisch, J. and Lingens, F. (1979) Biochem. J.. 181:771.

Chiariotti, L., Alifano, P., Carlogano, M. S. and Bruni, C. B. (1986)Mol. Gen. Genet., 203:382.

Furth, E. E., Thilly, W. G., Penman, B. W., Liber, H. L. and Rand, W. G.(1981) Analytical Biochem., 110:1-8.

Hansen, B. S., Vaughan, M. H. and Wang, L. J. (1972) J. Biol. Chem247:3854.

Kaufman, R. J. and Sharp, P. A. (1982) J. Mol. Biol., 159:601.

McKnight, S. L. and Gavis, E. R. (1980a) Nucl. Acids Res. 8, 5931-5948.

McKnight, S. L. (1980b) Nucl. Acids Res. 8, 5949-5964.

Mulligan, R. C. and Berg, P. (1985) Science 209:1422.

Southern, P. J. and Berg, P. (1982) J. Mol. Appl. Genet., 1:327.

Sugden, B., Marsh, K and Yates, J. (1985) Mol. Cell Biol. 5:410.

Wagner, M. J., Sharp, J. A. and Summers, W. C. (1981) Proc Natl. Acad.Sci. USA 78, 1441-1445.

Wigler, M. Silverstein, S., Lee, L. Pellicer, A. Cheng, T. and Axel, R.(1977) Cell 11:223.

Wigler, M , Sweet, R., Sim, G., Wold, B., Pellicer, A., Lacy, E.,Maniatis, T., Silverstein S. and Axel, R. (1979) Cell 16:777.

Yates, J. L., Warren, N and Sugden, B. (1985) Nature (Lond) 313:812.

Yoakum, G. H. (1984) Biotechniques 1/2:24.

What is claimed is:
 1. A method for selecting for mammalian cellscontaining a vector comprising,providing a vector carrying and capableof expressing in said mammalian cells a gene encoding histidinoldehydrogenase, introducing the vector in a mammalian cell line such thatthe encoded histidinol dehydrogenase is expressed, and selecting fromsaid cultured cells a cell that grows in the absence of agrowth-supporting, exogenous supply of histidine.
 2. A method as claimedin claim 1 further comprising growing said treated cells in mediumcontaining a precursor to histidine but not containing agrowth-supporting amount of histidine.
 3. A method as claimed in claim 2further comprising growing said treated cells in medium that containsthe precursor, wherein said precursor is histidine.
 4. A method forselecting transfected mammalian cells comprising,growing said mammaliancells transfected with a vector containing a gene encoding histidinoldehydrogenase, the histidinol dehydrogenase capable of effecting aprecursor to be converted ultimately into histidine, said growingcarried out in a medium supplemented with said precursor to histidine,but not in the presence of an exogenous supply of histidine sufficientto support growth.
 5. A preparation for use with mammalian cellscomprising,constituents present in relative amounts sufficient tosupport the growth of said mammalian cells, except that said preparationdoes not contain a growth-supporting amount of histidine and doescontain a precursor to histidine.
 6. A preparation as claimed in claim 5wherein said preparation is a solution selected from the groupconsisting of RPMI, MEM, DMEM and BME except that said solution containssaid precursor to histidine and does not contain a growth-supportingamount of histidine.
 7. A method as recited in any one of claims 1 or 4wherein the cells are human cells.